Method for preparing cell cultures from biological specimens for chemotherapeutic and other assays

ABSTRACT

An improved system for screening a multiple of candidate therapeutic or chemotherapeutic agents for efficacy as to a specific patient, in which a tissue sample from the patient is harvested, cultured and separately exposed to a plurality of treatments and/or therapeutic agents for the purpose of objectively identifying the best treatment or agent for the particular patient. Specific method innovations such as tissue sample preparation techniques render this method practically as well as theoretically useful. One particularly important tissue sample preparation technique is the initial preparation of cohesive multicellular particulates of the tissue sample, rather than enzymatically dissociated cell suspensions or preparations, for initial tissue culture monolayer preparation. With respect to the culturing of malignant cells, for example, it is believed (without any intention of being bound by the theory) that by maintaining the malignant cells within a multicellular particulate of the originating tissue, growth of the malignant cells themselves is facilitated versus the overgrowth of fibroblasts or other cells which tends to occur when suspended tumor cells are grown in culture. Practical monolayers of cells may thus be formed to enable meaningful screening of a plurality of treatments and/or agents. Growth of cells is monitored to ascertain the time to initiate the assay and to determine the growth rate of the cultured cells; sequence and timing of drug addition is also monitored and optimized. By subjecting uniform samples of cells to a wide variety of active agents (and concentrations thereof), the most promising agent and concentration for treatment of a particular patient can be determined. For assays concerning cancer treatment, a two-stage evaluation is contemplated in which both acute cytotoxic and longer term inhibitory effect of a given anti-cancer agent are investigated.

FIELD OF THE INVENTION

[0001] The invention relates to screening and testing of active agents,including chemotherapeutic agents, to predict potential efficacy inindividual patients in whom treatment with such agents is indicated.

INTRODUCTION

[0002] All active agents including chemotherapeutic active agents aresubjected to rigorous testing as to efficacy and safety prior toapproval for medical use in the United States. Methods of assessingefficacy have included elaborate investigations of large populations indouble blind studies as to a given treatment method and/or active agent,with concommitant statistical interpretation of the resulting data, butthese conclusions are inevitably generalized as to patient populationstaken as a whole. In many pharmaceutical disciplines and particularly inthe area of chemotherapy, however, the results of individual patienttherapy may not comport with generalized data—to the detriment of theindividual patient. The need has been long recognized for a method ofassessing the therapeutic potential of active agents, including but notlimited to chemotherapeutic agents, for their efficacy as to a givenindividual patient, prior to the treatment of that patient.

[0003] Prior art assays already exist which expose malignant tissue ofvarious types to a plurality of active agents, for the purpose ofassessing the best choice for therapeutic administration. For example,in Kruczynski, A., et al., “Evidence of a direct relationship betweenthe increase in the in vitro passage number of human non-small-cell-lungcancer primocultures and their chemosensitivity,” Anticancer Research,vol. 13, no. 2, pp. 507-513 (1993), chemosensitivity ofnon-small-cell-lung cancers was investigated in in vivo grafts, in invitro primocultures and in commercially available long-term cancer celllines. The increase in chemosensitivity was documented and correlatedwith morphological changes in the cells in question. Sometimes animalmodel malignant cells and/or established cell cultures are tested withprospective therapy agents, see for example Arnold, J. T., “Evaluationof chemopreventive agents in different mechanistic classes using a rattracheal epithelial cell culture transformation assay,” Cancer Res.,vol. 55, no. 3, pp. 537-543 (1995).

[0004] When actual patient cells are used to form in vitro assaysfocussed on individual patients, in typical prior art processes thecells are harvested (biopsied) and trypsinized (connective tissuedigested with the enzyme trypsin) to yield a cell suspension suitablefor conversion to the desired tissue culture form. The in vitro tissueculture cell collections which result from these techniques aregenerally plagued by their inability accurately to imitate thechemosensitivity of the original tumor or other cell biopsy. Standardcloning and tissue culture techniques are moreover excessivelycomplicated and expensive for use in a patient-by-patient assay setting.A need thus remains for a technique of tissue culture preparation whichprovides cell cultures, for drug screening purposes, in which aftersimple preparation the cell cultures react in a manner equivalent totheir in vivo reactivity, to enable drug or chemotherapeutic agentscreening as to a particular patient for whom such screening isindicated.

SUMMARY OF THE INVENTION

[0005] In order to meet this need, the present invention is an improvedsystem for screening a multiple of candidate therapeutic orchemotherapeutic agents for efficacy as to a specific patient, in whicha tissue sample from the patient is harvested, cultured and separatelyexposed to a plurality of treatments and/or therapeutic agents for thepurpose of objectively identifying the best treatment for the culturedcells obtained from the patient. Specific method innovations such astissue sample preparation techniques render this method practically aswell as theoretically useful. One particularly important tissue samplepreparation technique is the initial preparation of cohesivemulticellular particulates of the tissue sample, rather thanenzymatically dissociated cell suspensions or preparations, for initialtissue culture monolayer preparation. With respect to the culturing ofmalignant cells, for example, it is believed (without any intention ofbeing bound by the theory) that by maintaining the malignant cellswithin a multicellular particulate of the originating tissue, growth ofthe malignant cells themselves is facilitated versus the overgrowth offibroblasts or other cells which tends to occur when suspended tumorcells are grown in culture. Practical monolayers of cells may thus beformed to enable meaningful screening of a plurality of treatmentsand/or agents. Growth of cells is monitored to ascertain the time toinitiate the assay and to determine the growth rate of the culturedcells; sequence and timing of drug addition is also monitored andoptimized. By subjecting uniform samples of cells to a wide variety ofactive agents (and concentrations thereof), the most efficacious agentcan be determined. For assays concerning cancer treatment, a two-stageevaluation is contemplated in which both acute cytotoxic and longer terminhibitory effect of a given anti-cancer agent are investigated.

DETAILED DESCRIPTION OF THE INVENTION

[0006] The present invention is a system for screening a multiple ofcandidate therapeutic or chemotherapeutic agents for efficacy as to aspecific patient, in which a tissue sample from the patient is harvestedand separately exposed to a plurality of treatments and/or therapeuticagents for the purpose of objectively identifying the best treatment oragent. Specific method innovations such as tissue sample preparationtechniques render this method practically as well as theoreticallyuseful. One particularly important tissue sample preparation techniqueis the initial preparation of cohesive multicellular particulates(explants) of the tissue sample, rather than enzymatically dissociatedcell suspensions or preparations, for initial tissue culture monolayerpreparation. Cell growth, and sequence and timing of drug addition, aremonitored and optimized.

[0007] An important application of the present invention is thescreening of chemotherapeutic agents and other antineoplastic therapiesagainst tissue culture preparations of malignant cells from the patientsfrom whom malignant samples are biopsied. Related anti-cancer therapieswhich can be screened using the inventive system are both radiationtherapy and agents which enhance the cytotoxicity of radiation, as wellas immunotherapeutic anti-cancer agents. Screening processes fortreatments or therapeutic agents for nonmalignant syndromes are alsoembraced within this invention, however, and include without limitationagents which combat hyperproliferative syndromes, such as psoriasis, orwound healing agents. Nor is the present efficacy assay limited only tothe screening of active agents which speed up (healing) or slow down(anti-cancer, anti-hyperproliferative) cell growth because agentsintended to enhance or to subdue intracellular biochemical functions maybe tested in the present tissue culture system also. For example, theformation or blocking of enzymes, neurotransmitters and otherbiochemicals may be screened with the present assay methods prior totreatment of the patient.

[0008] When the patient is to be treated for the presence of tumor, inthe preferred embodiment of the present invention a tumor biopsy of>100mg of non-necrotic, non-contaminated tissue is harvested from thepatient by any suitable biopsy or surgical procedure known in the art.Biopsy sample preparation generally proceeds as follows under a LaminarFlow Hood which should be turned on at least 20 minutes before use.Reagent grade ethanol is used to wipe down the surface of the hood priorto beginning the sample preparation. The tumor is then removed, understerile conditions, from the shipping container and is minced withsterile scissors. If the specimen arrives already minced, the individualtumor pieces should be divided into four groups. Using sterile forceps,each undivided tissue quarter is then placed in 3 ml sterile growthmedium (Standard F-10 medium containing 17% calf serum and a standardamount of Penicillin and Streptomycin) and systematically minced byusing two sterile scalpels in a scissor-like motion, or mechanicallyequivalent manual or automated opposing incisor blades. Thiscross-cutting motion is important because the technique creates smoothcut edges on the resulting tumor multicellular particulates. Preferablybut not necessarily, the tumor particulates each measure 1 mm³. Aftereach tumor quarter has been minced, the particles are plated in cultureflasks using sterile pasteur pipettes (9 explants per T-25 or 20particulates per T-75 flask). Each flask is then labelled with thepatient's code, the date of explantation and any other distinguishingdata. The explants should be evenly distributed across the bottomsurface of the flask, with initial inverted incubation in a 37° C.incubator for 5-10 minutes, followed by addition of about 5-10 mlsterile growth medium and further incubation in the normal, non-invertedposition. Flasks are placed in a 35° C., non-CO₂ incubator. Flasksshould be checked daily for growth and contamination. Over a period of afew weeks, with weekly removal and replacement of 5 ml of growth medium,the explants will foster growth of cells into a monolayer. With respectto the culturing of malignant cells, it is believed (without anyintention of being bound by the theory) that by maintaining themalignant cells within a multicellular particulate of the originatingtissue, growth of the malignant cells themselves is facilitated versusthe overgrowth of fibroblasts (or other unwanted cells) which tends tooccur when suspended tumor cells are grown in culture.

[0009] The use of the above procedure to form a cell monolayer culturemaximizes the growth of malignant cells from the tissue sample, and thusoptimizes ensuing tissue culture assay of chemotherapeutic action ofvarious agents to be tested. Enhanced growth of actual malignant cellsis only one aspect of the present invention, however; another importantfeature is the growth rate monitoring system used to oversee growth ofthe monolayer once formed. Once a primary culture and its derivedsecondary monolayer tissue culture has been initiated, the growth of thecells is monitored to ascertain the time to initiate the chemotherapyassay and to determine the growth rate of the cultured cells.

[0010] Monitoring of the growth of cells is conducted by counting thecells in the monolayer on a periodic basis, without killing or stainingthe cells and without removing any cells from the culture flask. Thecounting may be done visually or by automated methods, either with orwithout the use of estimating techniques known in the art (counting in arepresentative area of a grid multiplied by number of grid areas, forexample). Data from periodic counting is then used to determine growthrates which may or may not be considered parallel to growth rates of thesame cells in vivo in the patient. If growth rate cycles can bedocumented, for example, then dosing of certain active agents can becustomized for the patient. The same growth rate can be used to evaluateradiation treatment periodicity, as well. It should be noted that withthe growth rate determinations conducted while the monolayers grow intheir flasks, the present method requires no hemocytometry, flowcytometry or use of microscope slides and staining, with all theirconcommitant labor and cost.

[0011] Protocols for monolayer growth rate generally use aphase-contrast inverted microscope to examine culture flasks incubatedin a 37° C. (5% CO₂) incubator. When the flask is placed under thephase-contrast inverted microscope, ten fields (areas on a grid inherentto the flask) are examined using the 10× objective, with the provisothat the ten fields should be non-contiguous, or significantly removedfrom one another, so that the ten fields are a representative samplingof the whole flask. Percentage cell occupancy for each field examined isnoted, and averaging of these percentages then provides an estimate ofoverall percent confluency in the cell culture. When patient sampleshave been divided between two or among three or more flasks, an averagecell count for the total patient sample should be calculated. Thecalculated average percent confluency should be entered into a processlog to enable compilation of data—and plotting of growth curves—overtime. Monolayer cultures may be photographed to document cell morphologyand culture growth patterns. The applicable formula is:${{Percent}\quad {confluency}} = \frac{{estimate}\quad {of}\quad {the}\quad {area}\quad {occupied}\quad {by}\quad {cells}}{{total}\quad {area}\quad {in}\quad {an}\quad {observed}\quad {field}}$

[0012] As an example, therefore, if the estimate of area occupied by thecells is 30% and the total area of the field is 100%, percent confluencyis 30/100, or 30.

[0013] Adaptation of the above protocol for non-tumor cells isstraightforward and generally constitutes an equivalent procedure.

[0014] Active agent screening using the cultured cells does not proceedin the initial incubation flask, but generally proceeds using platessuch as microtiter plates. The performance of the chemosensitivity assayused for screening purposes depends on the ability to deliver areproducible cell number to each row in a plate and/or a series ofplates, as well as the ability to achieve an even distribution of cellsthroughout a given well. The following procedure assures that cells arereproducibly transferred from flask to microtiter plates, and cells areevenly distributed across the surface of each well.

[0015] The first step in preparing the microtiter plates is, of course,preparing and monitoring the monolayer as described above. The followingprotocol is exemplary and susceptible of variation as will be apparentto one skilled in the art. Cells are removed from the culture flask anda cell pellet is prepared by centrifugation. The cell pellet derivedfrom the monolayer is then suspended in 5 ml of the growth medium andmixed in a conical tube with a vortex for 6 to 10 seconds. The tube isthen rocked back and forth 10 times. A 36 μl droplet from the center ofthe conical tube is pipetted onto one well of a 96 well plate. A freshpipette is then used to pipette a 36 μl aliquot of trypan blue solution,which is added to the same well, and the two droplets are mixed withrepeated pipette aspiration. The resulting admixture is then dividedbetween two hemocytometer chambers for examination using a standardlight microscope. Cells are counted in two out of four hemocytometerquadrants, under 10× magnification. Only those cells which have nottaken up the trypan blue dye are counted. This process is repeated forthe second counting chamber. An average cell count per chamber is thusdetermined. Using means known in the art, the quadrant count values arechecked, logged, multiplied by 10⁴ to give cells/ml, and the totalamount of fluid (growth medium) necessary to suspend remaining cellaliquots is calculated accordingly.

[0016] After the desired concentration of cells in medium has beendetermined, additional cell aliquots from the monolayer are suspended ingrowth medium via vortex and rocking and loaded into a Terasakidispenser known in the art. Aliquots of the prepared cell suspension aredelivered into the microtiter plates using Terasaki dispenser techniquesknown in the art. A plurality of plates may be prepared from a singlecell suspension as needed. Plates are then wrapped in sterile wet cottongauze and incubated in an incubator box by means known in the art.

[0017] After the microtiter plates have been prepared, exposure of thecells therein to active agent is conducted, according to the followingexemplary protocol. During this portion of the inventive assay, theappropriate amount of specific active agent is tranferred into themicrotiter plates prepared as described above. A general protocol, whichmay be adapted, follows. Each microtiter plate is unwrapped from its wetcotton gauze sponge and microscopically examined for cell adhesion.Control solution is dispensed into delineated rows of wells within thegrid in the microtiter plate, and appropriate aliquots of active agentto be tested are added to the remaining wells in the remaining rows.Ordinarily, sequentially increasing concentrations of the active agentbeing tested are administered into progressively higher numbered rows inthe plate. The plates are then rewrapped in their gauze and incubated inan incubator box at 37° C. under 5% CO₂. After a predefined exposuretime, the plates are unwrapped, blotted with sterile gauze to remove theagent, washed with Hank's Balance Salt Solution, flooded with growthmedium, and replaced in the incubator in an incubator box for apredefined time period, after which the plates may be fixed and stainedfor evaluation.

[0018] Fixing and staining may be conducted according to a number ofsuitable procedures; the following is representative. After removal ofthe plates from the incubator box, culture medium is poured off and theplates are flooded with Hank's Balance Salt Solution. After repeatedflooding (with agitation each time) the plates are then flooded withreagent grade ethanol for 2-5 minutes. The ethanol is then poured off.Staining is accomplished with approximately 5 ml of Giemsa Stain perplate, although volume is not critical and flooding is the goal. Giemsastain should be left in place 5 min.±30 seconds as timing influencesstaining intensity. The Giemsa stain is then poured off and the platesare dipped 3 times cold tap water in a beaker. The plates are theninverted, shaken vigorously, and air dried overnight (with plate lidsoff) on a rack on a laboratory bench. Cells per well are then countedmanually or by automated and/or computerized means, to derive dataregarding chemosensitivity of cells at various concentrations ofexposure. One particularly useful computer operating environment forcounting cells is the commercially available OPTIMATE compiler, which isdesigned to permit an optical counting function well suited tocomputerized cell counting procedures and subsequent calculations.

[0019] The above procedures do not change appreciably when cell growthpromoters are assayed rather than cell arresting agents such aschemotherapeutic agents. The present assay allows cell death or cellgrowth to be monitored with equal ease. In any case, optimization of useof the present system will involve the comparative testing of a varietyof candidate active agents, for selection of the best candidate forpatient treatment based upon the in vitro results. One particularlyadvantageous embodiment of the above described invention comprises atwo-stage assay for cytotoxicity followed by evaluation of longer-terminhibitory effect. Chemotherapeutic agents may thus be evaluatedseparately for both their direct chemotherapeutic effect as well as fortheir longer duration efficacy.

[0020] Identification of one or more active agents or chemotherapeuticagents is peripheral to the present invention, which is intended for theefficacy screening of any or all of them as to a given patient.Literally any active agent may be screened according to the presentinvention; listing exemplary active agents is thus omitted here.

[0021] The essence of the invention thus includes the important featureof the simplicity of the present system—cohesive multicellularparticulates of the patient tissue to be tested are used to form cellmonolayers; growth of those monolayers is monitored for accurateprediction of correlating growth of the same cells in vivo; anddiffering concentrations of a number of active agents may be tested forthe purpose of determining not only the most appropriate agent but themost appropriate concentration of that agent for actual patient exposure(according to the calculated cell growth rates). It is also important tonote, in the context of the invention, that the present system allows invitro tests to be conducted in suspensions of tissue culture monolayersgrown nutrient medium under fast conditions (a matter of weeks), ratherthan with single cell progeny produced by dilution cloning over longperiods of time. In some cases, the present invention is a two stageassay for both cytotoxicity and the longer-term growth inhibitory.

[0022] Although the present invention has been described with respect tospecific materials and methods above, the invention is only to beconsidered limited insofar as is set forth in the accompanying claims.

I claim:
 1. A method for assessing chemosensitivity of patient cellscomprising the steps of: a) harvesting a specimen of a patient's tissue,cells ascites, or effusion fluid; b) separating said specimen intomulticellular particulates; c) growing a tissue culture monolayer fromsaid cohesive multicellular particulates; d) inoculating cells from saidmonolayer into a plurality of segregated sites; and e) treating saidplurality of sites with at least one active agent, followed byassessment of chemosensitivity of the cells in said site to at least oneactive agent.
 2. The method according to claim 1 wherein step a) furthercomprises the step of a) preparing a specimen which was harvested from asample of patient tumor tissue;.
 3. The method according to claim 1wherein said plurality of segregated sites further comprises a platecontaining a plurality of wells therein.
 4. The method according toclaim 1 wherein step e) further comprises the step of: e) treating saidplurality of sites with a plurality of active agents at variedconcentrations, followed by assessment of optimal chemosensitivity withrespect to a single active agent at a single concentration.
 5. Themethod according to claim 1 wherein step e) further comprises the stepof: e) treating said plurality of sites with a plurality of activeagents over a length of time adequate to permit assessment of bothinitial cytotoxic effect and longer-term inhibitory effect of at leastone of said plurality of active agents.
 6. The method according to claim1 wherein the chemosensitivity assayed according to step e) isanti-cancer sensitivity.
 7. The method according to claim 1 wherein stepd) is accomplished using a Terasaki dispenser.
 8. The method accordingto claim 1 wherein the cells in step d) are prepared in suspension priorto inoculation into a plurality of wells in a culture plate.
 9. Themethod according to claim 1, wherein said active agent is achemotherapeutic agent.
 10. The method according to claim 1, whereinsaid active agent is a wound healing agent.
 11. The method according toclaim 1, wherein said active agent is a radiation therapy and/or aradiation therapy sensitizing or ameliorating agent.
 12. The methodaccording to claim 1, where said active agent is an immunotherapeuticagent.